The present application hereby claims priority under 35 U.S.C. 119 of the Japanese application No. 10/332760, filed Nov. 24, 1998.
The present invention relates to an antisense oligonucleotide against chromosomal DNA and/or RNA encoding CXCR4 protein and an HIV infection inhibitor containing the antisense oligonucleotide. More specifically, the present invention relates to an antisense oligonucleotide which can inhibit the HIV infection, by hybridizing specifically with the chromosomal DNA and/or RNA of the CXCR4 protein, being a receptor of the cell side concerning AIDS infection, to thereby inhibit the expression thereof, and an HIV infection inhibitor containing the antisense oligonucleotide.
The AIDS is a disease which is induced by human immunodeficiency virus (HIV) infection, injures cellular immunity greatly to thereby cause many kinds of opportunistic infections, lymphoma, neuropathy, or the like, and finally death certainly.
As the present remedy, a reverse transcriptase inhibitor such as azidothymidine (AZT), dideoxy inosine (ddI), dideoxy cytidine (ddC), or a protease inhibitor such as saquinavir, ritonavir, indinavir is used alone or in combination (Hammner, S. M. et al., New Engl. J. Med., 335, 1081-1090,1996). The nucleotide reverse transcriptase inhibitor inhibits the integration of virus into chromosome, by acting in the step when the reverse transcriptase of the virus itself transcripts the gene information of the virus from RNA into DNA after HIV enters cells. However, there is some case where resistant viruses are appeared by a long-term administration of these reverse transcriptase inhibitor and protease inhibitor, and thus the drugs become unavailable (Shirasaka, T. et al., Proc. Natl. Acad. Sci. USA, 92,2398-2402, 1995;Condra, J. H. et al., Nature, 374, 569-571, 1995). Further, since it causes disorders on DNA metabolism on the cell side, many side effects such as anemia, decrease of the number of leukocytes, nausea, head ache, malaise, stupor, myositis due to the long-term administration, have been reported. Thus developments of a new therapy have been desired strongly.
The main target cells of the HIV are CD4-positive T cell and macrophage. The HIV can be divided greatly into three kinds: a strain which infects CD4 positive T cell and does not infect macrophage (T cell tropic HIV), a strain which infects macrophage and does not infect CD4 positive T cell (macrophage tropic HIV), and a strain which can infect both cells (both tropic HIV) . From the past, as a receptor of the HIV on the cell side, CD4 has been known, but the second receptor concerning the T cell-tropic characteristics of the HIV was identified in 1996, and named CXCR4 (Feng, Y., et al., Science, 272, 872-877(1996)). In addition, at the same time, as a second receptor of the macrophage tropic HIV, CCR5 was identified (Alkhatib, G. et al., Science, 272, 1955-1958, 1996). These second receptors are indispensable factors on the cell side for HIV for infection as well as CD4, but these are primarily a receptor against chemokine secreted in living organisms. The fact that the ligand of the CXCR4 is SDF1, the ligand of CCR5 is MIP-1xcex1, MIP-1xcex2 and RANTES, are shown.
As to the therapy for HIV infectious diseases, many reports have been made from the past. However, since the mutation of genomic gene of HIV occurred in a very high rate compared with genomic gene of cell derived from mammal animals, there is a disadvantage that, even if a drug effective on HIV specifically is invented, the drug becomes ineffective on a mutated HIV. To the course of the study on the factors on the HIV side, some experiments have been made to treat the HIV infectious diseases by controlling the factors on the cell side concerning the HIV infections. With the method, since mutation of the genomoc gene of cells rarely occurs, there is a few possibility that the drug becomes unavailable by appearing a resistant virus which is shown in reverse transcriptase inhibitor. In addition, the conventional anti-HIV drug acts on virus itself after infecting on cells, on the other hand, this method inhibits the infection step of HIV, so that the HIV does not enter cells themselves, and there is a few possibility of injuring the viability and functions of the cell.
In recent years, many basic studies on the therapy of the HIV infectious disease directed to these second receptors, have been reported. For examples, the fact that SDF1, MIP-1xcex1, MIP-1xcex2 and RANTES inhibit the T cell tropic HIV and macrophage tropic HIV infections in a way of a competitive inhibition by competiting receptors (Bleul, C. C., et al., Nature, 829-833, 1996; Cocchi, F., et al., Science, 270,1811-1815, 1995), the fact that the antagonist inhibits the HIV infection, and a method of not expressing a receptor on the surface of a cell by expressing chemokine within a target cell excessively (Chen, J. D., et al., Nature Medicine, 3, 1110-1116, 1997), are considered. However, the large amount of chemokine administration stimulates the HIV infectious cells to thereby possibly release a large amount of HIV (Schmidtmayerova, H., et al., Nature, 382, 767, 1996). Thus when it is to be applied practically in therapy, there is a doubt in its effects.
The present invention was made in view of the above circumstances, and the object of the invention is to provide an antisense oligonucleotice which can inhibit the HIV infection to thereby prevent and treat the AIDS infection by inhibiting the CXCR4 protein expression of the target cell when infected with HIV, and an HIV infection inhibitor containing the antisense oligonucleotide.
The present inventors researched earnestly in order to dissolve the above problems, and as the results, succeeded in finding antisense gene sequences which inhibit specifically the CXCR4 protein expression to thereby inhibit the infection of HIV into cells, and completed the present invention.
Thus, the present inventors, as an antisense oligonucleotide inhibiting specifically the CXCR4 protein expression, considered the gene encoding the CXCR4 protein as a target gene, and examined repeatedly in view of the facts that the coding region, G cap region, initiation codon region or the like may possibly hybridize (Takeuchi, K., et al., Experimental Medicine, 573-583, 1996), and they selected the initiation codon region of the CXCR4 so as to complete the present invention.
Namely, the present invention comprises an antisense oligonucleotide characterized in that it hybridizes specifically with the chromosomal DNA and/or RNA encoding CXCR4 protein to thereby inhibit the CXCR4 protein expression, and it has one or more of the following sequences (A) to (C):
(A) the sequence described in the SEQ ID NO:1 in the SEQUENCE LISTING,
(B) the sequence described in the SEQ ID NO:2 in the SEQUENCE LISTING,
(C) the sequence described in the SEQ ID NO:3 in the SEQUENCE LISTING.
Further, the present invention comprises an HIV infection inhibitor containing the above antisense oligonucleotide.